Infectious disease remains a leading cause of mortality worldwide. A significant aspect of this problem is the continuing rise of infections that are resistant to most, if not all, conventional antibiotics. To meet this challenge it is essential that new drug targets be identified, and new classes of antibiotics developed. Over the past two decades a large number of naturally-occurring antimicrobial peptides have been found in both vertebrate and invertebrate species that are capable of providing a rapid and broad-spectrum response against a wide variety of pathogens. Because the specificity of these peptides is based on recognition of general properties of the cell membrane the emergence of resistance is exceedingly rare, making them ideal starting points for the development of new antibiotics. One limiting factor in our ability to further enhance the efficacy of these peptides is a lack of detailed knowledge about the manner in which they interact with and disrupt target cell membranes. The studies outlined in this proposal are directed towards developing a clear understanding of peptide-membrane interactions for a promising class of antimicrobial peptides that are synthetic hybrids of the insect peptide cecropin A and the bee-venom peptide, mellitin. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy provides a powerful and well-established approach for the analysis of peptide-membrane interactions that is uniquely suited to providing such a detailed understanding. Specifically, we will use both conventional and pulsed SDSL EPR to measure membrane binding affinities, determine structure, topology, and degree of membrane insertion for cecropin-mellitin hybrid peptides in model membranes that mimic both eukaryotic and bacterial membranes. These properties will be related to antibiotic efficacy against a panel of drug-sensitive and drug-resistant bacteria. We will systematically modify peptide composition to define relationships between sequence, membrane interactions, and antibacterial efficacy. Finally, we will synthesize and evaluate the antibiotic efficacy and membrane interactions of peptidomimetic analogs composed of non-natural 2-amino acids. These studies will significantly advance our understanding of the mechanism of action of antimicrobial peptides, and contribute to the further development of peptide and peptidomimetic antibiotics. PUBLIC HEALTH RELEVANCE: The prevalence of multi-drug resistant (MDR) infections is one of the most serious problems in health care today, both in the United States and worldwide, leading to increased treatment costs and a growing incidence of treatment failure. There is a critical need for the development of new antibiotics, and in particular for new classes of compounds that target non-traditional sites other than cell-wall synthesis and the bacterial ribosome. Antimicrobial peptides, which display remarkable efficacy against a broad spectrum of pathogens, including those resistant to conventional antibiotics, offer a novel approach to the treatment of drug-resistant infections. Developing a more complete understanding of the interactions of antimicrobial peptides with their target cells will enhance our ability to design and develop more effective peptide and peptidomimetic antibiotics.